The invention relates to human cells which are capable, on the basis of an activation of the endogenous human EPO gene, of producing EPO in sufficient amount and purity to permit economical preparation of human EPO as a pharmaceutical preparation. The invention furthermore relates to a method of preparing such human EPO-producing cells, DNA constructs for activating the endogenous EPO gene in human cells, and methods for the large-scale production of EPO in human cells.
Erythropoietin (EPO) is a human glycoprotein which stimulates the production of red blood cells. EPO occurs in the blood plasma of healthy persons only in very low concentrations, so that preparation in large amounts is not possible in this manner. EP-0148 605 and EP-B-0205 564 describe the preparation of recombinant human EPO in CHO cells. The EPO described in EP-B-0148 605 has a higher molecular weight than urinary EPO and no O-glycosylation. Meantime, the EPO described in EP-B-0 205 564 from CHO cells, is available in large amounts and in pure form, but it originates from nonhuman cells. Moreover, the ability of CHO cells to produce is often relatively limited.
Furthermore, the harvesting of human EPO from the urine of patients with aplastic anemia is known (Miyake et al., J. Biol. Chem. 252 (1977), 5558-5564). Therein a seven-step process is disclosed which includes ion exchanger chromatography, ethanol precipitation, gel filtration and adsorption chromatography. An EPO preparation with a specific activity of about 70,000 U/mg of proteins obtained in a 21% yield. Disadvantages of this process and other methods of obtaining urinary EPO consist in the procurement of starting material in sufficient amounts and in repeatable quality. Furthermore, the purification from urine is difficult and even a purified product is not free of urinary contaminants.
GB-A-2085 887 describes a method for the preparation of human lymphoblastoid cells which are capable of producing EPO in small amounts. The economical production of a pharmaceutical with these cells is not possible.
WO 91/06667 describes a method for the recombinant preparation of EPO. In a first process step in primary human embryo kidney cells the endogenous EPO gene is brought by homologous recombination into operable linkage with a viral promoter and the DNA is isolated from these cells. In a second step the DNA thus isolated is transformed into nonhuman CHO cells and the expression of EPO in these cells is analyzed. No mention is found that production of EPO in human cells is possible.
WO 93/09222 describes the production of EPO in human cells, wherein a relatively high EPO production of up to 960,620 mU/106 cells/24 h is found in human fibroblasts which had been transfected with a vector containing the complete EPO gene. These cells contain an exogenous EPO gene which is not at the correct EPO gene locus, so that problems are to be expected with regard to the stability of the cell line. No information on a constitutive EPO production is found in WO 93/092222. Furthermore, neither is there any information as to whether the EPO produced can be obtained in a quality sufficient for pharmaceutical purposes.
Furthermore, in WO 93/09222 an activation of the endogenous EPO gene in human HT1080 cells is described. In it an EPO production is found of only 2,500 mU/106 cells in 24 h (corresponding approximately to 16 ng/106 cells/24 h). Such low production is entirely unsuited for the economical production of a pharmaceutical preparation.
WO 94/12650 and WO 95/31560 describe how a human cell with an endogenous EPO gene activated by a viral promoter is able, after amplification of the endogenous EPO gene, to produce up to approximately 100,000 mU/106 cells/24 h (corresponding to about 0.6 xcexcg/106 cells/24 h) of EPO. This amount too is still not sufficient for the economical production of a pharmaceutical.
The problem on which the present invention is based thus consisted in eliminating at least partially the above-described disadvantage of the state of the art and to offer a technologically better method for the preparation of EPO in human cells. Especially, this is to make it possible to obtain a product in sufficient quantity and purity to permit economical production for pharmaceutical purposes.
This problem is solved by activation of the endogenous EPO gene in human cells and if desired by subsequent amplification of the activated human EPO gene. In this manner it has surprisingly been possible, by the selection of suitable starting cells, DNA constructs and selection strategies, to provide human cells which are capable of producing EPO in sufficient quantity, quality and purity to permit the economical production of pharmaceutical preparations. Especially after amplification of the activated endogenous EPO gene, cells can be obtained which have a definitely higher production output than the CHO production cells used previously for the preparation of recombinant EPO.
One subject matter of the invention is a human cell which contains a copy of an endogenous EPO gene in operable linkage with a heterologous expression control sequence active in the human cell and is capable without prior gene amplification of producing at least 200 ng of EPO/106 cells per 24 hours. Preferably the human cell according to the invention is capable of the production of 200 to 3000 ng EPO/106 cells/24 h, and most preferably for the production of 1000 to 3000 ng EPO/106 cells/24 h.
Another subject matter of the present invention is a human cell which is obtainable by gene amplification from the cell previously described and contains several copies of an endogenous EPO gene, each in operable linkage with a heterologous expression control sequence active in the human cell, and is capable of the production of at least 1,000 ng EPO/106 cells/24 h. With special preference the human cell line obtainable by gene amplification is capable of producing 1,000 to 25,000 ng EPO/106 cells/24 h, and most preferably for the production of 5,000 to 25,000 ng EPO/106 cells/24 h.
The human cell is any cell, provided it can be cultured in vitro. Especially preferred are human cells which can be cultured in a serum-free medium, and especially in suspension.
In this manner the production of EPO can be performed in a large fermenter with a culture capacity of, for example, 1,000 liters.
Especially preferred is a human cell which is an immortalized cell, for example an HT 1080 cell (Rasheed et al., Cancer 33 (1974), 1027-1033), a HeLa S3 cell (Puck et al., J. Exp. Med. 103 (1956), 273-284), a Namalwa cell (Nadkarni et al., Cancer 23 (1969), 64-79) or a cell derived therefrom. An example of a cell according to the invention is the clone xe2x80x9cAladinxe2x80x9d which was deposited on Jul. 15, 1997 according to the prescriptions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, 38124 Braunschweig, under number DSM ACC 2320.
In the cell according to the invention, the endogenous EPO gene is linked with a heterologous expression control sequence which is active in the human cell. The expression control sequence comprises a promoter and preferably additional expression-improving sequences, e.g., an enhancer. The promoter can be a inducible or a constitutive promoter. Preferably the promoter is a strong viral promoter, e.g., an SV40 or a CMV promoter. The CMV promoter/enhancer is especially preferred.
Furthermore, to optimize the EPO expression it may be preferred for the endogenous EPO gene in the human cell, which is in operable association with the heterologous promoter, to have a signal peptide-coding sequence which is different from the natural signal peptide-coding sequence and codes preferably for a signal peptide with a modified amino acid sequence. Especially preferred is a signal peptide-coding sequence which codes for a signal peptide sequence modified in the region of the first four amino acids which is selected from
Met-X1-X2-X3,
wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, on the condition that X1-X2-X3 is not the sequence Gly-Val-His, and especially from
(a) Met-Gly-Ala-His, (SEQ ID NO:8)
(b) Met-Ser-Ala-His SEQ ID NO:10
(c) Met-Gly-Val-Pro SEQ ID NO:12 or
(d) Met-Ser-Val-His SEQ ID NO:14.
It is especially preferred that the sequence of the first four amino acids of the signal peptide be Met-Ser-Ala-His SEQ ID NO:10.
In an additional aspect, the present invention relates to a method for preparing a human EPO-producing cell, as previously stated, comprising the steps:
(a) Preparing human starting cells which contain at least a copy of an endogenous EPO gene,
(b) Transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous with regions of the human EPO gene locus, to permit a homologous recombination,
(ii) a positive selection marker gene, and
(iii) a heterologous expression control sequence which is active in the human cell,
(c) Culturing the transfected cells under conditions which select for the presence of the positive selection marker gene,
(d) Analyzing the cells selectable according to step (c), and
(e) Identifying the EPO-producing cells.
The DNA construct used in preparing the human EPO-producing cell contains two flanking DNA sequences which are homologous with regions of the human EPO gene locus in order to permit a homologous recombination. The selection of suitable flanking sequences is performed, for example, by the methods described in WO 90/11 354 and WO 91/09 955. Preferably the flanking sequences have each a length of at least 150 bp. With special preference the homologous DNA sequences are selected from the region of the 5xe2x80x2-untranslated sequences, exon 1 and intron 1 of the EPO gene. It is especially preferred to use a DNA sequence modified in the region of exon 1, which codes for a signal peptide modified in the first amino acids. The modifications in the exon 1 region are preferably as stated above.
The selection marker gene can be any selection marker gene suitable for eucaryotic cells, which upon expression leads to a selectable phenotype, e.g., antibiotic resistance, auxotrophy, etc. An especially preferred positive selection marker gene is the neomycin phosphotransferase gene.
Optionally, a negative selection marker gene such as, e.g., the HSV-thymidine kinase gene, can also be present, by whose expression cells are destroyed in the presence of a selection agent.
If an amplification of the EPO gene endogenously activated in the human cell is desired, the DNA construct contains an amplification gene. Examples of suitable amplification genes are dihydrofolate reductase, adenosine deaminase, ornithine decarboxylase, etc. An especially preferred amplification gene is the dihydrofolate reductase gene, especially a dihydrofolate reductase arginine mutant which has a lower sensitivity to the selective agent (methotrexate) than the wild type gene (Simonsen et al., Proc. Natl. Acad. Sci. USA 80 (1983); 2495).
If an amplification gene is present in the DNA construct used for activating the EPO gene, the method of the invention can also comprise the following steps:
(f) Amplification of the DNA sequence coding for EPO, and
(g) Harvesting of EPO-producing cells which contain a number of copies, greater in comparison with the starting cell, of an endogenous DNA sequence coding formature EPO in operable linkage with a heterologous expression control sequence.
Appropriate DNA constructs for the activation of the endogenous EPO gene present in the human starting cell are the plasmids listed in the examples: p187, p189, p190 and p192. Especially preferred is the plasmid p189 which was deposited with the DSMZ in accord with the provisions of the Budapest Treaty on Jul. 16, 1997 under the accession number DSM 11661, or a plasmid derived therefrom. The DNA constructs are preferably circular plasmid molecules which are used for the transfection of the human cell in a linearized form.
An additional subject of the present invention is a DNA construct for the activation of an endogenous EPO gene in a human cell, comprising:
(i) two flanking DNA sequences which are chosen homologous to regions of the human EPO gene locus selected from 5xe2x80x2-untranslated sequences, exon 1 and intron 1, in order to permit a homologous recombination, a modified sequence being present in the exon 1 region coding for the amino acids:
Met-X1-X2-X3,
wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, on the condition that X1-X2-X3 is not the sequence Gly-Val-His, and especially for the amino acids:
(a) Met-Gly-Ala-His SEQ ID NO:8,
(b) Met-Ser-Ala-His SEQ ID NO:10,
(c) Met-Gly-Val-Pro SEQ ID NO:12 or
(d) Met-Ser-Val-His SEQ ID NO:14,
(ii) a positive selection marker gene,
(iii) a heterologous expression control sequence which is active in a human cell, and
(iv) in some cases an amplification gene.
Still another subject of the present invention is a DNA construct for the activation of an endogenous EPO gene in a human cell, comprising:
(i) two flanking DNA sequences which are homologous to regions of the human EPO gene locus, selected from 5xe2x80x2-untranslated sequences, exon 1 and intron 1, to permit a homologous recombination,
(ii) a positive selection marker gene,
(iii) a heterologous expression control sequence which is active in a human cell, the distance between the heterologous expression control sequence and the translation start of the EPO gene being no greater than 1100 bp, and
(iv) in some cases, an amplification gene.
Surprisingly it was found that, in the case of a modification of the EPO signal sequence and/or a shortening of the distance between the heterologous expression control sequence and the translation start of the EPO gene, an optimized expression is obtained. Preferably the distance between the promoter of the heterologous expression control sequence and the translation start of the EPO gene is not greater than 1100 bp, especially preferred is not more than 150 bp, and most preferably not more than 100 bp. An especially preferred example of a DNA construct to be used according to the invention is the plasmid p189 (DSM 11661) or a plasmid derived therefrom.
Still another aspect of the present invention is a method for preparing human EPO, wherein a human cell according to the invention is cultured in a suitable medium under conditions in which a production of EPO takes place and the EPO is harvested from the culture medium. A serum-free medium is used preferentially. The cells are preferably cultured in suspension. More preferred, the culturing is performed in a fermenter, especially in a large fermenter with a capacity of, for example, 10 l-50,000 l.
The harvesting of human EPO from the culture medium of human cell lines comprises preferably the following steps:
(a) Passing the cell supernatant over an affinity chromatography medium and harvesting the fractions containing EPO,
(b) optionally, passing the EPO-containing fractions over a hydrophobic interaction chromatography medium and harvesting the EPO-containing fractions,
(c) passing the EPO-containing fractions over hydroxyapatite and harvesting the EPO-containing fractions, and
(d) concentrating and/or passing over a reverse-phase (RP) HPLC medium.
Step (a) of the purification process includes passing the cell supernatant, which in some cases can be pre-treated, over an affinity chromatography medium. Preferred affinity chromatography media are those to which a blue dye is coupled. An especially preferred example is blue sepharose.
After elution from the affinity chromatography medium the EPO-containing eluate is passed, in some cases, over a hydrophobic interaction chromatography medium. This step is expedient if a culture medium with a serum contentxe2x89xa72% (v/v) is used. If a culture medium with a low serum content is used, e.g., 1% (v/v), or a serum-free medium is used, this step can be omitted. A preferred hydrophobic interaction chromatography medium is butyl-sepharose.
The eluate from step (a) or, if used, step (b), is passed in step (c) of the method of the invention over hydroxyapatite and the EPO-containing eluate is subjected to a concentrating step and/or a reverse-phase HPLC purification step. The concentration is performed preferably by exclusion chromatography, e.g., membrane filtration, the use of a medium, such as a membrane with an exclusion size of 10 kD, having proven desirable.
By the method according to the invention an isolated human EPO with a specific activity of at least 100,000 U/mg protein in vivo (normocythemic mouse) is obtainable, which is free from urinary contaminants and can differ in its glycosylation from recombinant EPO from CHO cells. Preferably, the EPO of the invention has a specific activity of at least 175,000, and with special preference at least 200,000 to 400,000 or 450,000 IU/mg protein. The human EPO obtainable by the method of the invention can contain xcex1-2,3- and/or xcex1-2,6-linked sialic acid residues. In studies of EPO from cells which contain an endogenously activated EPO gene, on the basis of the present preliminary results, the presence of xcex1-2,3- and xcex1-2, 6-linked sialic acid residues were found. Furthermore, it was found on the basis of the present preliminary results that the human EPO of the invention has a content of less than 0.2% N-glycol neuraminic acid, with respect to the content of N-acetyl neuraminic acid.
The purity of the human EPO of the invention amounts preferably to at least 90%, more preferably at least 95%, and most preferably at least 98% of the total protein content. The determination of the total protein content can be performed by reverse phase HPLC, e.g., with a Poros R/2H column.
Furthermore, by the method of the invention, human EPO species are obtain able which differ in their amino acid sequence. Thus it was found by mass spectrometric analysis (MALDI-MS) that a human EPO can be isolated from HeLa S3 cells, which is mainly a polypeptide with a length of 165 amino acids, which is formed by C-terminal processing of an arginine residue, and in some cases includes up to 15% of an EPO with 166 amino acids. Also, a human EPO is obtainable which includes a polypeptide with a length of 166 amino acids, i.e., a non-processed EPO. From Namalwa cells, for example, a human EPO has been isolated which comprises a mixture of polypeptides with a length of 165 and 166 amino acids.
This human EPO can be used as an active substance for a pharmaceutical preparation which can contain additional active substances, as well as pharmaceutically common adjuvants, vehicles and additives.
In still another aspect the present invention relates to an isolated DNA which codes for a human EPO with a sequence modified in the region of the first four amino acids of the signal peptide, which is selected from:
Met-X1-X2-X3
wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, on the condition that X1-X2-X3 is not the sequence Gly-Val-His, and especially for the amino acids:
(a) Met-Gly-Ala-His SEQ ID NO:8,
(b) Met-Ser-Ala-His SEQ ID NO:10
(c) Met-Gly-Val-Pro SEQ ID NO:12 or
(d) Met-Ser-Val-His SEQ ID NO:14.
The DNA can be for example a genomic DNA or a cDNA.